Ca++-triggered synchronized release of the neurotransmitter at the synapse, which underlies neuronal communication and synaptic plasticity, requires membrane fusion. This remarkable process is controlled by an exquisitely orchestrated array of protein-protein interactions. SNARE (soluble N- ethylmaleimide-sensitive factor attachment protein receptor) assembly is the central event that may drive membrane fusion. A Ca++-sensor synaptotagmin I (SytI) and complexins impinge upon SNARE assembly to control the timing and the size of the release. The present project uses the newly developed single fusion assay based on total internal reflection (TIRF) microscopy to investigate the mechanism by which SNARE-dependent fusion is regulated by SytI, complexins, and Ca++. The single fusion assay makes it possible to dissect and characterize individual fusion intermediates in unprecedented detail. Further, this technique allows us to track the dynamic transitions in a single fusion event with msec time resolution. With this powerful new fusion assay, we will dissect how the fusion regulators modulate the individual fusion steps. Further, the present project uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), an established technique for the investigation of structures and topologies of membrane proteins. On the basis of various EPR results, a structural model of the protein in the native- like phospholipid bilayer is generated at the backbone resolution. In this project, conformational changes of SNARE complexes induced by SytI, complexins, and Ca++ are determined using SDSL EPR to gain insights into the structural basis of the synchronized release. The combined approach employing the single fusion assay and SDSL EPR will provide insights into mechanism whereby the synchronized release is manufactured in the neuron. Dysfunction of synchronous neurotransmitter release is linked to hideous mental illnesses such as schizophrenia, autism, and epilepsy, as well as to less serious illnesses including behavioral disorders. The outcomes of the proposed research will lead to the understanding of the mechanism whereby the synchronous release is controlled, which will eventually lead to the mechanism-based design of drugs for metal illnesses.